Tricyclic compounds having antimitotic and/or antitumor activity and methods of use thereof

ABSTRACT

The present invention provides tricyclic compounds, and pharmaceutically acceptable salts thereof, having antimitotic activity, anti-multidrug resistance activity, for example P-glycoprotein inhibition, and antitumor activity, and which inhibit paclitaxel sensitive and resistant tumor cells. Also provided are methods of utilizing these compounds for treating tumor cells and inhibiting mitosis of cancerous cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional utility patent application claimingpriority to U.S. patent application Ser. No. 15/795,801, filed on Oct.27, 2017, which is a divisional patent application claiming priority toU.S. patent application Ser. No. 15/283,705, filed on Oct. 3, 2016, nowU.S. Pat. No. 9,802,955, granted Oct. 31, 2017, which is a divisionalpatent application claiming priority to U.S. patent application Ser. No.14/719,824, filed on May 22, 2015, now U.S. Pat. No. 9,458,167, grantedOct. 4, 2016, which is a divisional patent application claiming priorityto U.S. patent application Ser. No. 13/151,536, filed Jun. 2, 2011, nowU.S. Pat. No. 9,056,869, granted Jun. 16, 2015, which is a continuationutility patent application claiming priority to U.S. patent applicationSer. No. 12/170,633, filed on Jul. 10, 2008, now U.S. Pat. No.7,982,035, granted Jul. 19, 2011, which is a continuation-in-partapplication claiming priority to U.S. patent application Ser. No.11/845,143, filed Aug. 27, 2007, now U.S. Pat. No. 7,960,400, grantedJun. 14, 2011, having the same named inventor, Aleem Gangjee. Thecontents of U.S. patent application Ser. Nos. 11/845,143, 12/170,633,13/151,536, 14/719,824, 15/283,705, and 15/795,801 are incorporated byreference in their entireties herein.

FIELD OF THE INVENTION

The present invention relates to tricyclic heteroaromatic compounds andtheir methods of use and, more particularly, to tricyclic heteroaromaticcompounds that are antitumor agents that inhibit the function ofmicrotubules (antimitotic agents or mitotic inhibitors) and that haveantitumor activity. These tricyclic heteroaromatic compounds inhibitP-glycoprotein (Pgp) infected tumor cells, and inhibit paclitaxelsensitive and resistant tumor cells. The compounds may be made into acidsalts that are water soluble for providing orally active antitumoragents.

BACKGROUND OF THE INVENTION

Mitosis is the process of nuclear division in eukaryotic cells thatproduces two daughter cells from one parent cell. The daughter cells andthe original parent cell have identical chromosomes and DNA. Generally,cancer is a disease of mitosis. It is believed that cancer begins when asingle cell is converted from a normal cell to a cancer cell. This isoften due to a change in function of one or more genes that normallyfunction to control cell growth. The cancer cells proliferate byrepeated and uncontrolled mitosis, in contrast to normal cells whichundergo only about 20 to 50 generations of replication and then cease. Atumor may be thought of a mass of unhealthy cells that are dividing andgrowing in an uncontrolled way.

Microtubules are long, protein polymers that are hollow, tube-likefilaments found in certain cell components such as the mitotic spindle.Each microtubule is composed of repeating subunits of the proteintubulin. Microtubules aggregate to form spindle fibers. During mitosis,cells use their spindle fibers to line up chromosomes, make copies ofthem, and divide into new cells with each new daughter cell having asingle set of chromosomes. The polymerization dynamics of microtubulesplay a pivotal role in this process as part of cell replication. Thecrucial involvement of microtubules in mitosis makes them a target forantitumor agents. Antitumor agents that inhibit the function ofmicrotubules are known as antimitotic agents.

Many classes of antimitotic agents are known. One such class is thevinca alkaloids exemplified by vincristine, vinblastine, vindesine, andvinorelbine. The vinca alkaloids are used in the treatment of leukemias,lymphomas, and small cell lung cancer. Another class of antimitoticagents are the taxanes, exemplified by paclitaxel (commerciallyavailable from Bristol-Myers Squibb Company under the tradename TAXOL)and docetaxel. The taxanes are useful in the treatment of breast, lung,ovarian, head and neck, and bladder carcinomas. Colchicine typifiesanother class of antimitotic agents. Colchicine, while not used as anantitumor agent, is a microtubule polymerization inhibitor. Lastly, thecombrestatins are another class of antitumor agents. Antimitotic agentssuch as the vinca alakaloids, colchicine, colcemid, and nocadazol blockmitosis by keeping the mitotic spindle from being formed. These agentsbind to the tubulin and inhibit polymerization, preventing cells frommaking the spindles they need to move chromosomes around as they divide.In contrast, paclitaxel binds to the tubulin protein of microtubules,locking the microtubules in place and inhibiting their depolymerization.With the mitotic spindle still in place, a cell may not divide intodaughter cells.

Multidrug or multiple drug resistance (MDR) is a major drawback ofcancer chemotherapy. Ultimate failure of chemotherapy often times occurswith the use of antimitotic agents due to MDR. MDR may be inherentlyexpressed by some tumor types while others acquire MDR after exposure tochemotherapy. P-glycoprotein (Pgp) is a 170 kilodalton (kDa) proteinthat belongs to the ATP-binding cassette superfamily of transporters.Pgp has been implicated as a primary cause of MDR in tumors. Pgps areefflux transporters found in the gut, gonads, kidneys, biliary system,brain, and other organs. A series of homologous proteins termedmultidrug-resistance proteins (MRPs) are also known. MRPs are associatedwith MDR in tumors. The first MRP termed MRP1 was identified in a drugresistant lung cancer cell line that expressed Pgp. All of thesetransporters bind drugs within cells and release them to theextracellular space using ATP. Tumor cells pre-exposed to cytotoxiccompounds often allow the cells to manifest resistance in the presenceof the cytotoxic drug. Overexpression of Pgp has been reported in anumber of tumor types, particularly after the patient has receivedchemotherapy, indicating the clinical importance of Pgp in MDR. Theclinical significance of Pgp along with its limited expression in normaltissues makes Pgp a viable target for inhibition to reverse MDR.

While antimitotic agents have shown to be some of the most successfulagents against malignancies, resistance, both intrinsic and acquired,often results in treatment failures. Thus, there exists a need todevelop new compounds that possess antimitotic activity, anti-multidrugresistance activity, and antitumor activity, that may be used alone as asingle agent in the treatment of cancer, or in combination withchemotherapeutic agents, including antimitotic agents, that shallinhibit mitosis in a wide variety of cells, including cells that aresubject to MDR. There is a need, therefore, for single compounds whichprovide the desired antimitotic, anti-multidrug resistance and antitumoractivities with a high degree of selectivity and low toxicity, and thatare effective inhibitors of paclitaxel sensitive and resistant tumorcells.

SUMMARY OF THE INVENTION

The present invention meets the above need by providing tricycliccompounds having antimitotic activity, anti-multidrug resistanceactivity (for example, Pgp inhibition), and antitumor activity in asingle molecule so that significant drawbacks of different aspects ofdrug transport of two or more drugs to their targets, additive orsynergistic toxicities of two or more different drugs, resistance ofcancer cells to a particular drug, as well as the cost associated withtwo or more drugs, is circumvented.

The present invention provides single compounds that exhibit antimitoticactivity, anti-multidrug resistance activity (for example, Pgpinhibition), and antitumor activity in tumor cells, such as, withoutlimitation, leukemia, non-small cell lung cancer, colon cancer, centralnervous system cancer, melanoma, ovarian cancer, renal cancer, prostatecancer and breast cancer; and other proliferative diseases anddisorders.

The present invention provides single compounds having a combinatorialchemotherapeutic potential of antimitotic activity, anti-multidrugresistance activity, and antitumor activity, and which inhibitpaclitaxel sensitive and resistant tumor cells.

In an aspect of the present invention, there is provided a compound ofFormula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent. Preferably, the compound ofFormula I, as described herein, comprises pharmaceutically acceptablesalts, prodrugs, solvates, or hydrates thereof.

In another embodiment of this invention the compound of Formula I, asdescribed herein, further comprises wherein when the C ring is saturatedor partially saturated, the substituted R₄ creates chirality when X is aC and R₆ and R₇ are different, then all stereoisomers thereof bothseparately and as racemic and/or diastereoisomeric mixtures areincluded.

In another aspect of the present invention, there is provided a compoundof Formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero). Preferably,the compound of Formula II, as described herein, comprisespharmaceutically acceptable salts, prodrugs, solvates, or hydratesthereof.

In another embodiment of this invention the compound having Formula II,as described herein, further comprises wherein when the C ring issaturated or partially saturated, the substituted R₄ creates chiralitywhen X is a C and R₆ and R₇ are different, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula I, asdescribed herein, or the salt, prodrug, solvate, or hydrate thereof.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula II, asdescribed herein, or the salt, prodrug, solvate, or hydrate thereof.

In yet another embodiment of this invention, a method for inhibiting themitosis of one or more cancerous cells is provided comprising subjectingone or more live cancerous cells to a mitotic inhibitory amount of acompound of Formula I, as described herein, or a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate of the compound of FormulaI, for effecting the inhibition of mitosis of the cancerous cell(s).

Another embodiment of this invention provides a method for inhibitingthe mitosis of one or more cancerous cells comprising subjecting atleast one live cancerous cells to a mitotic inhibitory amount of acompound of Formula II, as described herein, or a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate of a compound of FormulaII, for effecting the inhibition of mitosis of the cancerous cell(s).

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention can be gained from the followingdescription of the preferred embodiments when read in conjunction withthe accompanying drawings in which:

FIG. 1 show an immunofluorescence assay of A10 rat smooth muscle tumorcell line before treatment with a compound of the present invention(i.e. control).

FIG. 2 shows a microtubule depolymerization immunofluorescence assay ofA10 rat smooth muscle tumor cell line following treatment with acompound of the present invention, namely, Sample ID AAG3.

FIG. 3 shows the chemical structures of two compounds of the presentinvention, namely, Sample ID AAG3 and AAG 13.

FIG. 4 shows the biological effects of the compounds of the presentinvention.

FIG. 5a shows the results of the National Cancer Institute's preclinicalin vitro tumor screening panel evaluating a compound of the presentinvention, namely, Sample ID AAG3.

FIG. 5b shows the results of the National Cancer Institute's preclinicalin vitro tumor screening panel evaluating a compound of the presentinvention, namely, Sample ID AAG3.

FIG. 6a shows individual dose response curves of percentage growth forthe cancer cell lines set forth in FIG. 5.

FIG. 6b shows individual dose response curves of percentage growth forthe cancer cell lines set forth in FIG. 5.

FIG. 6c shows individual dose response curves of percentage growth forthe cancer cell lines set forth in FIG. 5.

FIG. 7 shows a dose response curve of percentage growth for all of thecell lines shown in FIG. 5.

FIG. 8a shows mean graphs for specific cancer types and correspondingcell lines shown in FIG. 5.

FIG. 8b shows mean graphs for specific cancer types and correspondingcell lines shown in FIG. 5.

FIG. 8c shows mean graphs for specific cancer types and correspondingcell lines shown in FIG. 5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides tricyclic compounds having antimitoticactivity, anti-multidrug resistance activity (for example, Pgpinhibition), and antitumor activity, and which inhibit paclitaxelsensitive and resistant tumor cells, in a single molecule and methods ofuse thereof.

The present invention provides a compound of the Formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero.)

In another embodiment of this invention the compound having Formula I,as described herein, further comprises wherein when the C ring issaturated or partially saturated, the substituted R₄ creates chiralitywhen X is a C and R₆ and R₇ are different, then all stereoisomersthereof both separately and as a racemic and/or diastereoisomericmixture(s) are included.

Compounds of the present invention, as described herein, may contain anasymmetric center and may thus exist as enantiomers. Where the compoundsaccording to the present invention posses two or more asymmetriccenters, they may additionally exist as diastereomers. The presentinvention includes all such possible stereoisomers as substantially pureresolved enantiomers, racemic mixtures thereof, and as mixtures ofdiastereomers. The formulas as described herein are shown without regardto a definitive stereochemistry at certain positions. The presentinvention includes all stereoisomers of such formulae andpharmaceutically acceptable salts thereof. Diastereoisomeric pairs ofenantiomers may be separated by methods known by those skilled in theart, for example, fractional crystallization from a suitable solvent,and the pair of enantiomers thus obtained may be separated intoindividual stereoisomers by conventional means, known by those skilledin the art, for example by the use of an optically active acid or baseas a resolving agent or on a chiral HPLC column. Further, any enantiomeror diastereomer of a compound of the formulae of the present inventionmay be obtained by stereospecific synthesis using optically purestarting materials or reagents of known configuration.

Preferably, the compounds of Formula I, as described herein, arepharmaceutically acceptable salts, prodrugs, solvates, or hydrates ofthe compound of Formula I.

Another embodiment of this invention provides a compound of Formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero). Preferably,the compounds of Formula II are provided as pharmaceutically acceptablesalts, prodrugs, solvates or hydrates thereof.

In another embodiment of this invention the compound having Formula II,as described herein, further comprises wherein when the C ring issaturated or partially saturated, the substituted R₄ creates chiralitywhen X is a C and R₆ and R₇ are different, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included.

Other embodiments of the present invention provide pharmaceuticallyacceptable salts, solvates, and hydrates of the compounds of Formulae Iand II. Preferably, the compounds of the present invention representedby Formulae I and II may be made into acid salts that are water soluble.Most preferably, these water soluble salts of Formulae I and II may beformulated into an oral dosage forms providing orally administeredactive antitumor agents. In the past, antimitotic agents have beenplagued with water solubility problems, such as for example but notlimited to Taxol® and combrestastatin, and a variety of solubilizingagents have been employed to improve their water solubility. The presentsalts of Formulae I and II overcome such water solubility problems andare generally completely water soluble.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula I, or apharmaceutical acceptable salt, prodrug, solvate, or hydrate of thecompound of Formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero).

As used herein, the term “patient” means members of the animal kingdom,including, but not limited to, human beings. As used herein, the term“having cancer” means that the patient has been diagnosed with cancer.

As used herein, the term “therapeutically effective amount” refers tothat amount of any of the present compounds required to bring about adesired effect in a patient. The desired effect will vary depending onthe illness being treated. For example, the desired effect may bereducing tumor size, destroying cancerous cells, and/or preventingmetastasis, any one of which may be the desired therapeutic response. Onits most basic level, a therapeutically effective amount is that amountneeded to inhibit the mitosis of a cancerous cell or to facilitate thereversal of multidrug resistance, particularly, for example due toP-glycoprotein, (i.e. an effective mitotic inhibitory amount). Anyamount of mitotic inhibition or reversal of multidrug resistance willyield a benefit to a patient and is therefore within the scope of theinvention.

In another embodiment of this invention the method of treating a patienthaving cancer by administering to the patient a therapeuticallyeffective amount of compound of Formula I, as described herein, furthercomprises wherein the compound of Formula I wherein the C ring issaturated or partially saturated, the substituted R₄ creates chiralitywhen X is a C and R₆ and R₇ are different, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula II, or apharmaceutical acceptable salt, prodrug, solvate, or hydrate of thecompound of Formula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero).

In another embodiment of the method for treating a patient having cancerby administering to the patient a therapeutically effective amount of acompound of the Formula II further comprises wherein the compound ofFormula II wherein the C ring is saturated or partially saturated, thesubstituted R₄ creates chirality when X is a C and R₆ and R₇ aredifferent, then all stereoisomers thereof both separately and as racemicand/or diastereoisomeric mixtures are included.

Compounds of the present invention covered under Formula I and II mayalso be administered with one or more additional treatment agents, i.e.,a chemotherapeutic agent. Suitable candidates for the additionalchemotherapeutic agent include for example but are not limited to,paclitaxel, docetaxel, vinca alkaloids, colchicines, colcemid,cisplatin, and nocadazol. The presence of the compound of the presentinvention shall enhance the effectiveness of the chemotherapeutic agentby facilitating the reversal of multidrug resistance, particularly dueto Pgp, and at least partially restoring the sensitivity of tumors toantimitotic agents.

In yet another embodiment of this invention, a method for inhibiting themitosis of one or more cancerous cells is provided comprising subjectingone or more live cancerous cells to an effective inhibitory amount of acompound of Formula I, or a salt, prodrug, solvate, or hydrate of acompound of Formula I:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero), for effectingthe inhibition of mitosis of the cancerous cells.

Another embodiment of this invention provides a method for inhibitingthe mitosis of one or more cancerous cells comprising subjecting livecancerous cells to an effective mitotic inhibitory amount of a compoundof Formula II, or a salt, prodrug, solvate, or hydrate of a compound ofFormula II:

wherein both B and C rings may be completely or partially saturated orunsaturated with respect to bond 4a-8a, 5-6 and 7-8; the C ring may havean R₄ attached to the C ring at positions 5, 6, 7 or 8, or a combinationof one or more of these positions depending on the saturation level ofthe C ring and wherein R₄ may be the same or different when attached toa plurality of the 5, 6, 7, or 8 positions of the C ring;

R₁ and R₂ each independently comprise one of (a) a hydrogen (H), (b) analkyl having from one to ten carbon atoms and having a straight orbranched configuration, and wherein the alkyl is partially or completelysaturated, or a substituted alkyl having from one to ten carbon atoms,(c) a cycloalkyl having from three to ten carbon atoms, or a substitutedcycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

R₁ and R₂ are the same or different with the exception that R₁ and R₂may not each be hydrogen at the same time;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

X comprises one of (a) a NH, (b) a NR₅, (c) an oxygen (O), (d) a sulfur(S), (e) a CR₅, and (f) a CR₅R₁₀, wherein R₅ and R₁₀ may be the same ordifferent and comprise one of R₁; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero), for effectingthe inhibition of mitosis of the cancerous cells.

As used herein, the term “lower alkyl” group refers to those lower alkylgroups having one to about ten carbon atoms, such as for example methyl,ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,cyclohexyl, cyclopropylmethyl or cyclobutylmethyl groups. Alkyl groupssharing one to about six carbon atoms are preferred. These lower alkylgroups are straight chain, branched chain or cyclic (alicyclichydrocarbon) arrangements. The carbon atoms of these straight chain,branched chain or cyclic arranged alkyl groups may have one or moresubstituents for the hydrogens attached to the carbon atoms.

As used herein, the term “heteroalkyl” refers to alkyl chains from oneto about 3 atoms where one or more of the carbons has been replaced withnitrogen, oxygen or sulfur, Thus “heteroalkyl” groups will include, forexample, —CH₂—CH₂—NH—, —CH₂—S—, —S—CH₂—, —CH₂—O—, —CH₂—CH₂—O—, —O—CH₂—,—NH—CH₂—CH₂₋, —N—CH⁼CH—, and other various combinations, as will beapparent to one skilled in the art. The above list is not meant to beexhaustive, and many combinations are contemplated as within the scopeof the present invention.

The term “aryl” groups, as used herein, refers to compounds whosemolecules have an aromatic ring structure, such as the six-carbon ringof benzene, or multiple rings which are either fused or unfused, such ascondensed six-carbon rings of other aromatic derivatives. The term“aryl” is also defined to include diaryl, triaryl and polyaryl groups,which would have two, three or more rings, respectively. Thus, suitablearyl groups would include, for example, phenyl, biphenyl, naphthyl,phenanthrene, anthracene groups and aryl oxyaryl groups. This list isnot meant to be exhaustive, and any aryl group, as these terms aredefined above and commonly understood in the art, are within the scopeof the present invention.

The term “heteroaryl” refers to aromatic ring structures having at leastone atom in the ring which is not carbon, such as oxygen, nitrogen orsulfur. “Heteroaryls” as used herein also refers to aromatic ringstructures that are part of larger ring structures, such as two or threemember ring systems, which may be fused or unfused, in which one of therings is as described above. Thus, “heteroaryl” refers to ring systemsin which one or more rings contain a heteroatom and one or more rings donot. It will be understood that this list is not meant to be exhaustive,and that any heteroaryl group, as these terms are defined above andcommonly understood in the art, are within the scope of the presentinvention. The heteroaryl ring systems may be fused ring systems orunfused. Examples of heteroaryl ring systems include, for example butare not limited to, pyridine, quinoline, isoquinoloine, pyrrole,thiophenes, furans, imidazoles, and the like, as well as fused ringstructures having rings of different sizes, such as benzofurans,indoles, purines, and the like.

Also included within the scope of the present invention are alicyclicgroups, as that term is understood in the art, and heterocyclic groups.As used herein, the term “heterocyclic group” refers to non-aromaticcyclic substituents in which one or more members of the ring is notcarbon, and is at least one of an oxygen, sulfur or nitrogen atom, forexample.

The terms “alkylaryl” (or “alkaryl”) or “alkylheteroaryl” as used hereinrefer to groups having an alkyl moiety attached to an aryl or heteroarylring. The alkyl moiety is preferably a straight, branched or cyclicalkyl group having one to about six carbon atoms. This alkyl moiety mayalso contain oxygen, nitrogen or sulfur, and therefore may be an alkoxygroup. The aryl or heteroaryl moiety of the alkylaryl group is asubstituted or unsubstituted aryl or heteroaryl group, as these termsare described above. As used herein, the terms “alkylaryl” or“alkylheteroaryl” will also be used to refer to arylalkyl groups orheteroarylalkyl groups, as those terms are understood in the art, anddenotes attachment of such a substituent at either the alkyl or the arylportion of the group. Thus, for example, a benzyl group would beembraced by the term “alkylaryl”.

Any of the cyclic substituents described above, such as the aryl,heteroaryl, alkylaryl, alkylheteroaryl, alicyclic, or heterocyclicgroups are optionally substituted with one or more substituents aslisted above. In the case of more than one substituent, the substituentsare independently selected. “Alkoxy groups” and “alkyl groups” includestraight or branched chains having up to about ten members. “Halogen”refers to chlorine, bromine, iodine and fluorine. “Aryl and heteroarylgroups” are as described above. When a carboxylic acid is a substituent,it will be appreciated that the moiety represents an acid such asbenzoic acid.

As used herein, the terms “aroyl” or “heteroaroyl”, such as when usedwithin the term p-aroyl-L-glutamate, refers to benzoyl, napthoyl,thiophenoyl, furophenoyl, pyrroyl, and any other “aroyl” or“heteroaroyl” as these terms would be understood by one skilled in theart. “Aroyl” and “heteroaroyl” are generally defined in the art as anaromatic or heteroaromatic compound having a carbonyl moiety. As usedherein, the term “glutamate” will be understood as representing both theester form (glutamate) and the acid form (glutamic acid).

It will appreciated by those skilled in the art that a general formuladepicting compounds having side chains with adjacent carbons having adouble bond will result in both cis and trans isomers as possiblestructures. Both the cis and trans isomers, and mixtures thereof, of anysuch compound within the broad general formula described in Formulas Iand II are contemplated as being within the scope of the presentinvention.

A preferred form of Formula I is shown in FIG. 3, Sample ID AAG3.

Proliferative diseases and/or disorders that may be treated according tothe methods of the present invention include, without limitation,leukemia, non-small cell lung cancer, colon cancer, central nervoussystem (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, and breast cancer.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a tricycliccompound of the present invention to produce the desired effect inassociation with a pharmaceutical carrier. The specification for thedosage unit forms of the invention are dictated by and directlydependent on the particular compound and the particular effect, ortherapeutic response, that is desired to be achieved.

Compounds containing Formula I or Formula II, or pharmaceuticallyacceptable salts, prodrugs, solvates, or hydrates thereof, can beadministered to a patient (an animal or human) via various routesincluding parenterally, orally or intraperitoneally. Parenteraladministration includes the following routes that are outside thealimentary canal (digestive tract): intravenous; intramuscular;interstitial, intraarterial; subcutaneous; intraocular; intracranial;intraventricular; intrasynovial; transepithelial, including transdermal,pulmonary via inhalation, ophthalmic, sublingual and buccal; topical,including dermal, ocular, rectal, or nasal inhalation via insufflationor nebulization. Specific modes of administration shall depend on theindication. The selection of the specific route of administration andthe dose regimen is to be adjusted or titrated by the clinicianaccording to methods known to the clinician in order to obtain theoptimal clinical response. The amount of compound to be administered isthat amount which is therapeutically effective. The dosage to beadministered to a patient shall depend on the characteristics of thepatient being treated, including for example, but not limited to, thepatient's age, weight, health, and types and frequency of concurrenttreatment, if any, of any other chemotherapeutic agent(s), all of whichis determined by the clinician as one skilled in the art.

Compounds containing Formula I or Formula II, or a salt, a prodrug, asolvate, or a hydrate thereof, that are orally administered can beenclosed in hard or soft shell gelatin capsules, or compressed intotablets. Compounds also can be incorporated with an excipient and usedin the form of ingestible tablets, buccal tablets, troches, capsules,sachets, lozenges, elixirs, suspensions, syrups, wafers and the like.Compounds containing Formula I or Formula II can be in the form of apowder or granule, a solution or suspension in an aqueous liquid ornon-aqueous liquid, or in an oil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch; gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoring.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic. Additionally, thecompounds of Formula I or Formula II, and a salt thereof, can beincorporated into sustained-release preparations and formulations.

The compounds of Formula I or Formula II, or a salt, a prodrug, asolvate, or a hydrate thereof, can be administered to the centralnervous system, parenterally or intraperitoneally. Solutions of thecompound as a free base or a pharmaceutically acceptable salt can beprepared in water mixed with a suitable surfactant, such ashydroxypropylcellulose. Dispersions also can be prepared in glycerol,liquid polyethylene glycols and mixtures thereof, and in oils. Underordinary conditions of storage and use, these preparations can contain apreservative and/or antioxidants to prevent the growth of microorganismsor chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compounds of the present invention may be contained within, mixed with,or associated with, a suitable (acceptable) pharmaceutical carrier foradministration to a patient according to the particular route ofadministration desired. Suitable or acceptable pharmaceutical carriersrefer to any pharmaceutical carrier that will solubilize the compoundsof the present invention and that will not give rise to incompatabilityproblems, and includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such suitable or acceptablepharmaceutical carriers are well known by those skilled in the art.Preferred carriers include sterile water, physiologic saline, and fivepercent dextrose in water. Examples of other suitable or acceptablepharmaceutical carriers include, but are not limited to, ethanol, polyol(such as propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, or vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size (in the case of adispersion) and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the compoundof Formula I or Formula II in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the sterilized compound of Formula I orFormula. II into a sterile vehicle that contains the basic dispersionmedium and any of the other ingredients from those enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of Formula I and Formula II, or a salt, aprodrug, a solvate, or a hydrate thereof, can be administered to apatient alone or in combination with pharmaceutically acceptablecarriers or as pharmaceutically acceptable salts, the proportion ofwhich is determined by the solubility and chemical nature of thecompound, chosen route of administration to the patient and standardpharmaceutical practice.

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLES

Flow cytometric analysis was performed to assess the effect of thetricyclic compounds of the present invention on the cell cycle phasedistributions of MDA MB 435 human breast cancer. The percentage of cellsin the G₂/M phases was increased approximately two-fold by treatment ofthe cells for twenty four hours with tricyclic compounds AAG3 and AAG13.

FIG. 1 shows a microtubule immunofluorescence assay of a A-10 rat smoothmuscle cell line before treatment with a compound of this invention(i.e. control). FIG. 2 shows the microtubule depolymerizationimmunofluorescence assay of an A-10 rat smooth muscle cell linefollowing treatment with the compound of the present invention AAG3.AAG3 has structural Formula I wherein R₁ is a methoxybenzyl group and R₂and R₃ are each a methyl group and wherein ring C is saturated, andwherein X is an O. A-10 rat smooth muscle cells were used since theygrow as flat monolayers that are amenable to imaging. The A-10 cellswere treated for twenty four (24) hours (h) with Et0H (control), and250nM (nanomolar) AAG3. Microtubules were then visualized by indirectimmunofluorescence staining with beta-tubulin antibodies. The controlcells shown in FIG. 1 displayed extensive microtubule systems withperimeter organizing centers. Treatment with AAG3 caused losses ofmicrotubules in the cells. This immunofluorescence assay of FIG. 2 showsthat AAG3 was effective in depolymerizing the tubulin proteinmicrotubule of A-10 cells. The AAG3 compound has potent nanomolartubulin inhibitory activity. Compounds of the present invention, havingthe structural Formulae I and II, as set forth herein, inhibit themicrotubule dynamics. The inhibition of microtubule dynamics hindersmicrotubule formation and results in mitotic arrest and initiation ofapoptosis or programmed cell death.

The biological effects of two of the compounds of the present invention,namely AAG3 and AAG13, as compared to known antimitotic agents Taxol®(Bristol-Myers Squibb Company) and combrestastatin A4, commerciallyavailable from Cayman Chemicals, Michigan, USA, are presented in FIG. 4.Antimitotic compounds AAG3, AAG 13, Taxol®, and combrestastatinA4, wereevaluated for cytotoxity towards the panel of human cell lines MDA MB435 (human breast cancer), SKOV3 (human ovarian cancer), and SKOV3M6/6(Pgp infected human ovarian cancer). FIG. 4 shows the IC₅₀ of each ofthese antimitotic compounds towards each cancer cell line. The IC₅₀ isthe inhibitory concentration required to effectuate fifty percentinhibition of cell growth. FIG. 4 shows that the compounds of thepresent invention, AAG3 and AAG13, have cytotoxic activity toward eachof the human cancer cell lines tested. Although Taxol® andcombrestastatin A4 were more potent than compound AAG3 in the MDA MB 435and the SKOV3 sensitive cell lines, Taxol® was subject to tumorresistance due to the overexpression of P-glycoprotein (Pgp) in theovarian cancer cell line SKOV3M6/6. FIG. 4 shows the IC50 values of 3.5nanoM (nM) for AAG3 and 4.4 microM (μM) for Taxol® toward the Pgpinfected human ovarian cancer cell line SKOV3M6/6. FIG. 4 shows thecalculated relative resistance value of 1 for compound

AAG3 and a relative resistance value of 2013 for Taxol®. Thus, theresults confirm that overexpression of Pgp did not effect cellsensitivity to compound AAG3 of the present invention.

FIG. 5 shows the results of testing compound AAG3 (otherwise identifiedas XZ/AG/153-423 (73100)) of the present invention using National cancerInstitute (NCI) 55 human tumor lines. The cells lines, which representleukemia, non-small cell lung cancer, colon cancer, central nervoussystem cancer, melanoma, ovarian cancer, renal cancer, prostate cancer,and breast cancer, are listed in FIG. 5. Testing was in accordance withthe NCI Developmental Therapeutics Program (DTP) In Vitro Cell LineScreening Project (IVCLSP). Methodology for testing under IVCLSP isprovided at http://dtp.nci.gov/branches/btb/ivclsp.html.

FIG. 5 shows the tumor cell inhibitory activity, measured by GI₅₀ values(10⁻⁸ M) for AAG3. GI₅₀ is the concentration of chemical required toreduce the growth of treated cells to half that of untreated cells (i.e.control). GI₅₀ represents the concentration of chemical required toeffectuate fifty percent inhibition of cell growth. AAG3 exhibited GI₅₀values of single digit 10⁻⁸ molar levels against all 55 tumor celllines.

An NCI COMPARE analysis was performed for AAG3 to elucidate a possiblemechanism of action by comparing responses of the 55 cell lines to knownmicrotubule-targeting agents. For microtubule specific compounds, thecell type selectivity profile in tumor growth inhibitory (TGI) levels ishighly indicative of the compound's mechanism of action. A TGICorrelation value that is equal to or greater than 0.6 is generallyconsidered by those skilled in the art to be a good correlation valuefor classification as a microtubule targeting agent. The results of theNCI COMPARE analysis for compound AAG3 of the present invention is setforth in Table 1.

TABLE 1 TGI endpoint TARGET SET: STANDARD_AGENTS_TGI SEED: S747156-4MTGI 2 days AVGDATA SEED TYPE: NSC_FIVE_DOSE Correla- Cell Rank Vectortion line 1 vincristine sulfate S67574-3M TGI 2 days 0.675 49 AVGDATA 2maytansine S 153858-4M TGI 2 days 0.6 49 AVGDATA 3 vinblastine sulfateS49842-5.6M TGI 2 days 0.53 49 AVGDATA 4 rhizoxin S332598-4M TGI 2 daysAVGDATA 0.52 49 5 rhizoxin S332598-4M TGI 2 days AVGDATA 0.467 47The NCI COMPARE analysis was performed for AAG3 to elucidate a possiblemechanism of action of AAG3 by the similarity response of the cell linesto known compounds. The four compounds that showed the best correlationwith AAG3 are all well-known microtubule targeting agents. Formicrotubule specific compounds, the cell type selectivity profile in TGI(Total Growth Inhibition) level is highly indicative of the compoundsmechanism of action. Thus AAG3 is a microtubule inhibitor. This COMPAREanalysis also indicates that AAG3 acts most like vincristine sulfate(correlation 0.7), which is a well known anticancer agent widely used inthe clinic and strongly suggests that AAG3 would be highly active invivo. The tumor inhibitory data from the NCI preclinical tumor screenalso strongly suggest in vivo activity for AAG3.

FIGS. 6a, 6b, and 6c show individual dose response curves of percentagegrowth for each of the cancer cell lines set forth in FIG. 5.

FIG. 7 shows a dose response curve of percentage growth for all of thecell lines shown in FIG. 5.

FIGS. 8a, 8b, and 8c show mean graphs for specific cancer types andcorresponding cell lines shown in FIG. 5.

Synthesis of Tricyclic Compounds

Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-DRY dryingapparatus over P₂O₅ at 80° C. Melting points were determined on aMEL-TEMP II melting point apparatus with FLUKE 51 K/J electronicthermometer and are uncorrected. Nuclear magnetic resonance spectra forproton (¹H NMR) were recorded on a Bruker WH-400 (400 MHz) spectrometer.The chemical shift values are expressed in ppm (parts per million)relative to tetramethylsilane as an internal standard: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet.Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254silica gel plates with a fluorescent indicator, and the spots werevisualized under 254 and 366 nm illumination. Proportions of solventsused for TLC are by volume. Column chromatography was performed on a230-400 mesh silica gel (Fisher, Somerville, N.J.) column. Elementalanalyses were performed by Atlantic Microlab, Inc., Norcross, Ga.Element compositions are within ±0.4% of the calculated values.Fractional moles of water or organic solvents frequently found in someanalytical samples could not be prevented in spite of 24-48 h of dryingin vacuo and were confirmed where possible by their presence in the ¹HNMR spectra. All solvents and chemicals were purchased from AldrichChemical Co. or Fisher Scientific and were used as received.

Synthesis of AAG3

Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-DRY dryingapparatus over P₂O₅ at 80° C. Melting points were determined on aMEL-TEMP II melting point apparatus with FLUKE 51 K/J electronicthermometer and are uncorrected. Nuclear magnetic resonance spectra forproton (¹H NMR) were recorded on a Bruker WH-400 (400 MHz) spectrometer.The chemical shift values are expressed in ppm (parts per million)relative to tetramethylsilane as an internal standard: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet.Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254silica gel plates with a fluorescent indicator, and the spots werevisualized under 254 and 366 nm illumination. Proportions of solventsused for TLC are by volume. Column chromatography was performed on a230-400 mesh silica gel (Fisher, Somerville, N.J.) column. Elementalanalyses were performed by Atlantic Microlab, Inc., Norcross, Ga.Element compositions are within ±0.4% of the calculated values.Fractional moles of water or organic solvents frequently found in someanalytical samples could not be prevented in spite of 24-48 h of dryingin vacuo and were confirmed where possible by their presence in the ¹HNMR spectra. All solvents and chemicals were purchased from AldrichChemical Co. or Fisher Scientific and were used as received.

Synthesis of AAG3

Chemistry:

Condensation between dihydroxyl pyrimidine 1 (Scheme 1) andα-Cl-cyclohexanone in basic condition afforded tricyclicfuro[2,3-d]pyrimidine 3. Solvent selection was very important for theyield of this reaction. Different solvent including DMF, DMSO, H₂O,MeOH/H₂O, EtOH/H₂O and ^(i)PrOH/H₂O were tried. Among them ^(i)PrOH/H₂Oas solvent gave the best result. Compound 3 was then treated with POCl₃to afford 4-cholo analogue 4, which reacted with N-methyl aniline 5 anda trace amount of HCl in BuOH to give the target compound AAG3.

Experimental section for Scheme 1:

2-Methyl-5,6,7,8-tetrahydro[1]benzofuro[2,3-d]pyrimidin-4(3H)-one (3).NaOH (0.4 g, 1 mmol) and compound 1 (1.26 g, 1 mmol) were dissolved 10mL H₂O. Compound 2 (1.32 g, 1 mmol) was dissolved in 10 mL ^(i)PrOH.After the two solutions were mixed in a 50 mL flask and refluxed for 3days, the reaction mixture was evaporated to dryness under reducedpressure. The residue was dissolved in a minimum amount of MeOH, andchromatographed on a silica gel column (2 cm×15 cm) with acetyl acetateas the eluent. Fractions that showed the desired single spot atR_(f)=0.55 were pooled and evaporated to dryness to afford 857 mg (42%)of 3 as a white powder: TLC R_(f) 0.35 (CHCl₃/MeOH 6:1); mp>300° C.; ¹HNMR (DMSO-d₆) δ 1.70 (q, 2 H, J=8 Hz), 1.79 (q, 2 H, J=8 Hz), 2.32 (s, 3H), 2.58 (d, 4 H, J=8 Hz), 12.29 (s, 1 H). Anal. calcd for C₁₁H₁₂N₂O₂:C, 64.69; H, 5.92; N, 13.72. Found: C, 64.54; H, 5.97; N, 13.43.

4-Chloro-2-methyl-5,6,7,8-tetrahydro[1]benzofuro[2,3-d]pyrimidine (4).To a 50 mL flask was added 3 (2.04 g, 10 mmol) and 10 mL POCl₃. Theresulting mixture was refluxed for 2 h, and the solvent was removedunder reduced pressure to afford a dark residue. To this was added 30 mLof CHCl₃ and 3 g of silica gel. The solvent was evaporated to afford aplug. Column chromatography of the plug with hexane: acetyl acetate=20:1as eluent afford 1.67 g (82%) of 4 as a yellow solid: TLC R_(f) 0.41(Hexane/EtOAc 15:1); mp 42.4-43.8° C.; ¹H NMR (DMSO-d₆) δ 1.79 (q, 2 H,J=8 Hz), 1.87 (q, 2 H, J=8 Hz), 2.63 (s, 3 H), 2.75 (d, 4 H, J=8 Hz).

N-(4-methoxyphenyl)-N,2-dimethyl-5,6,7,8-tetrahydro[1]benzofuro[2,3-d]pyrimidin-4-amine(AAG3). To a 50 mL flask was added 4(111 mg, 0.5 mmol), 5 (77 mg, 0.55mmol) and 5 mL BuOH. To this solution was added 2 drops of concentrateHCl solution and the mixture was refluxed. TLC indicated thedisappearance of starting material 4, the solvent was removed underreduced pressure. To the residue obtained was added silica gel and MeOHand the solvent removed to make a plug. This plug was separated bycolumn chromatography to give 117 g (73%) of AGG3 as white solid; TLCR_(f) 0.45 (Hexane/EtOAc 3:1); mp 147-149° C.; ¹H NMR (DMSO-d₆) δ 1.19(s, 3 H), 1.31 (m, 2 H), 1.57 (m, 2 H), 2.57 (s, 3 H), 3.26 (m, 2 H),3.39 (m, 2 H), 3.75 (s, 3 H), 6.96 (d, 2 H, J=3 Hz), 7.16 (d, 2 H, J=3Hz) Anal. calcd for: C, 70.57; H, 6.55; N, 12.99. Found. C, 70.63; H,6.56; N, 12.72.

Synthesis of AAG13

Chemical Discussion

The synthesis of target compounds commenced from commercially available1,2-dichloro-3-nitro-benzene 3 and ethyl cyanoacetate 2 (Scheme 2).After treating 2 with base potassium tert-butoxide, 3 was added to thereaction mixture. Displacement of the chloro group of 3 by the ethylcyanoacetate anion provided compound 4 as a viscous yellow liquid.Reduction of the nitro group of 4 followed by cyclization furnishedcompound 5 as a pink solid. Use of fresh or activated zinc powder isrecommended for this reaction. Cyclocondensation of 5 with carbamimidicchloride hydrochloride 6 afforded the tricyclic compound 8 as a brownsolid (synthesis of 7 is provided in scheme 2a). Protection of the2-amino group of 8 using pivalic anhydride under basic conditionsprovided 9. Compound 10 was prepared by treating 9 with phosphoryltrichloride at reflux. Replacement of the 4-chloro group in 10 with a4-methoxy-N-methyl aniline group, and simultaneous deprotection of the2-amino group in 10 was achieved in one step by treating 10 with4-methoxy-N-methyl aniline in IPA at reflux, in presence of 4 drops ofconcentrated hydrochloric acid, to obtain the target compound AAG13 in69% yield.

Experimental Section for Scheme 2 and 2a:

Ethyl(2-chloro-2-nitrophenyl)(cyano)acetate (4). To an ice cold solutionof ethylcyanoacetate (10.9 mL, 102.4 mmol) in anhydrous THF (170 mL)under nitrogen, was added potassium tert-butoxide (12.7 g, 107.5 mmol).The formed white suspension was stirred for 15 min, then treated with 2,3-dichloronitrobenzene (9.83 g, 51.2 mmol). The suspension was heated atreflux for 48 hours. The resulting reddish brown solution was poured into water, and the aqueous-mixture was acidified to pH 2 with conc. HCl.The mixture was extracted with ether (3×150 mL) and then the combinedorganic phase was dried (using Na₂SO₄) and concentrated to give a darkoil. Flash chromatography using 10:1 hexane:ethyl acetate in a columnpacked with silica gel, 10 times the weight of the dark oil, provided aviscous yellow liquid 4, which was used without further purification forthe next step. TLC Rƒ0.23 (hexane-ethyl acetate 3:1). ¹H NMR δ 1.33-1.38(t, 3H, CH₃); 4.29-4.35 (q, 2H, CH₂); 7.59-8.14 (m, 3H, phenyl).

Ethyl-2-amino-4-chloro-1H-indole-3-carboxylate (5). 4 (18 g, 67 mmol) in250 mL glacial acetic acid, was treated with a single charge of 18 g ofzinc dust. The mixture was heated at 55° C. for 45 minutes. Later 6 gmore zinc dust was added. After heating for another 105 minutes, theyellow mixture was filtered through a pad of celite. The pad was washedwith acetic acid and the filtrate was concentrated to a residue that wasdistributed between chloroform and water. The organic phase was washedwith NaHCO₃ (5%) to provide a pink precipitate which was filtered, driedover P₂O₅, dissolved in methanol, added silica gel and converted to asilica gel plug by removing the solvent under reduced pressure. The plugwas transferred on top of a column packed with silica gel, ten times theweight of plug, eluted with hexane, chloroform, 5% ethylacetate inchloroform and 10% ethylacetate in chloroform. Fractions containing theproduct 5 (TLC) were pooled and evaporated to give a pink solid. Theoverall yield from 3 to 5 was 63%. TLC Rƒ0.187 (hexane-chloroform 1:1);mp 140-142° C.; ¹H NMR (DMSO-d6) δ 1.25-1.28 (t, 3H, CH₃); 4.18-4.20 (q,2H, CH₂); 6.85 (bs, 2H, 2-NH₂, exch); 6.92-7.09 (m, 3H, phenyl); 10.93(bs, 1H, 9-NH, exch). Anal. Calculated (C₁₁H₁₁ClN₂O₂): C, 55.36; H,4.65; N, 11.74; Cl, 14.85. Found: C, 55.39; H, 4.60; N, 11.65; Cl,14.96.

Carbamimidic chloride hydrochloride (7). Cyanamide (4.2 g, 0.1 mol) wasdissolved in 100 mL of diethyl ether in a 500 mL round bottom flask. Themixture was stirred under nitrogen. 100 mL of 2M HCl in diethyl etherwas added to the reaction flask via a 250 mL dropping funnel. Stirringwas continued for 2 hours at room temperature. The white salt whichprecipitated out was filtered and dried. The overall yield for 7 was96%. Compound 7 was used for the next step without further purification.

2-Amino-5-chloro-3, 9-dihydro-4H-pyrimido[4,5-b]indol-4-one (8). Methylsulfone (1 g) was heated to melting. Compound 7 (106.22 mg, 1.37 mmol)was added and the resulting mixture was stirred and heated at 110-120°C. to dissolve completely. 5 (200 mg, 0.837 mmol) was added in one partto the reaction mixture. Stirring was continued for 30 minutes. About 10mL water was added to quench the reaction. Ammonia water was added toneutralize the reaction mixture. Solid precipitated out. This solid wasfiltered. Obtained solid was dissolved in chloroform and methanol, dried(using Na₂SO₄) and recrystallized. The overall yield was 78%. TLC Rƒ0.33(chloroform-methanol 1:1); mp>250° C.; ¹H NMR (DMSO-d6) δ 6.57 (bs, 2H,2-NH₂ exch); 7.04-7.17 (m, 3H, phenyl); 10.41 (s, 1H, 9-NH, exch); 11.64(s, 1H, 3-NH, exch). Anal. Calculated (C₁₀H₇ClN₄O. 0.3CH₃OH): C, 50.65;H, 3.38; N, 22.94; Cl, 14.52. Found: C, 50.91; H, 3.34; N, 22.60; Cl,14.77.

N-(5-chloro-4-oxo-4,9-dihydro-3H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide (9). Compound 8 (300 mg, 1.27 mmol), 2-dimethyl propanoicanhydride (713.32 mg, 3.83 mmol), dimethyl aminopyridine (7 mg, 0.06mmol), triethylamine (514.05 mg, 5.08 mmol) were weighed together in a50 mL round bottom flask. This flask was placed in an oil bath at 60° C.with stirring for 2 days. Then, to the reaction mixture was added 1 gsilica gel. The DMF was removed using oil pump and a silica gel plug wasmade. The plug was transferred on top of a column packed with silicagel, twenty times the weight of plug, eluted with chloroform, 1%methanol in chloroform and 5% methanol in chloroform. Fractionscontaining the product 9 (TLC) were pooled and evaporated to give solidcompound. The overall yield was 40%. TLC Rƒ0.45 (chloroform-methanol10:1), m.p. 185.8-190.1° C., ¹H NMR: δ 1.27 (s, 9H, pivaloyl); 7.19-7.40(m, 3H, phenyl); 11.15 (s, 1H, 9-NH, exch); 11.94 (s, 1H, 9-NH, exch);12.12 (s, 1H, 3-NH, exch).

N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethyl propanamide(10). To 9 (2 g, 6.274 mmol) was added 30 mL of POCl₃ in a 250 mL roundbottom flask. The reaction mixture was refluxed at 110-120° C. for 4hours. After this the POCl₃ was evaporated and the mixture wasneutralized using NH₄OH. The aqueous mixture was filtered (the ppt beingthe compound). The filtrate too contained some compound. Therefore itwas extracted using chloroform and ethyl acetate. The ppt. obtained wasdissolved in chloroform and methanol. Both the dissolved ppt. andextracted filtrate were dried using sodium sulfate overnight. To thesolution was added silica and solvent was removed under reduced pressureto provide a silica gel plug. The plug was transferred on top of acolumn packed with silica gel, twenty times the weight of plug, elutedwith chloroform, 1% methanol in chloroform and 5% methanol inchloroform. Fractions containing the product 10 (TLC) were pooled andevaporated to give a solid. The overall yield was 70%. TLC Rƒ0.86(chloroform-methanol 5:1), m.p. 245.6-246.1° C., ¹H NMR: δ 1.24 (s, 9H,pivaloyl); 7.37-7.63 (m, 3H, phenyl); 10.32 (s, 1H, 9-NH, exch); 12.96(s, 1H, 2-NH, exch). Anal. Calculated (C₁₅H₁₄Cl₂N₄O. 0.25 (C₂H₅)₂O): C,54.02; H, 4.67; N, 15.74. Found: C, 54.14; H, 4.56; N, 15.69.

5-Chloro-N⁴-(4-methoxyphenyl)-N⁴-methyl-9H-pyrimido[4,5-b]indole-2,4-diamineAAG13. To 10 (20 mg, 0.06 mmol) was added 4-methoxy-N-methyl aniline (61mg, 0.44 mmol), 50 mL of Isopropanol, and 4 drops of conc. HCl in a 100mL round bottom flask. The reaction mixture was refluxed at 110-120° C.for 26 hours, following which the Isopropanol was evaporated and themixture was basified with ammonia in methanol to obtain a slightprecipitate. The resulting precipitate was then dissolved in acetone andmethanol. To the solution was added silica, and the solvent was removedunder reduced pressure to provide a silica gel plug. The plug wastransferred on top of a column packed with silica gel, twenty times theweight of plug, eluted with chloroform and 1% methanol in chloroform.Fractions containing the product AAG13 (TLC) were pooled and evaporatedto give a solid which was further purified by washing with hexane. Theoverall yield was 69%. TLC Rƒ0.25 (chloroform-methanol 20:1), m.p.163-164° C., ¹H NMR: δ 3.30 (s, 3H, OCH₃), 3.66 (s, 3H, N—CH₃), 6.55 (s,2H, NH₂, exch), 6.76-7.25 (m, 7H, ArH), 11.65 (s, 1H, 9-NH, exch). Anal.Calculated (C₁₈H₁₆ClN₅O. 0.2 CH₃(CH₂)₄CH₃): C, 62.15; H, 5.10; N, 18.87;Cl, 9.55. Found: C, 61.79; H, 4.95; N, 18.72; Cl, 9.48.

Synthesis of AAG101

Chemistry:

The Gewald reaction of 1, 2 and 3 afforded bicyclic 4 (Scheme 3).Condensation of 4 and CH₃CN under acidic conditions afforded 5, whichwas converted to the 4-chloro analogue 6 by treating with POCl₃. The4-chloro compound 6 reacted with N-methyl aniline 7 and a trace amountof HCl in BuOH to give compound AAG101. Condensation between 4 and 9afforded tricyclic compound 10, which was converted to 4-chloro analogue11 by treating with POCl₃. The 4-chloro compound 11 reacted withN-methyl aniline 7 and a trace amount of HCl in BuOH to give compound12.

Experimental Section for Scheme 3

Ethyl 2-amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (4). Amixture of 1 (98 mg, 1 mmol), 2 (32 mg, 1 mmol), 3 (113 mg, 1 mmol) andEtOH (5 mL) was treated dropwise with morpholine (86 mg, 1 mmol) at 45°C. over 15 min. The mixture was stirred for 5 h at 45° C. and 24 h atroom temperature. Unreacted sulfur was removed by filtration, and thefiltration was concentrated under reduced pressure to afford an orangeoil. The residue was loaded on a column packed with silica gel andwashed with 10% ethyl acetate in hexane. The fractions containing thedesired product (TLC) were pooled and evaporated to afford 184 mg (82%)of 4 as a white solid:

2-Methyl-5,6,7,8-tetrahydro [1]benzothieno[2,3-d]pyrimidin-4(3H)-one(5). To a 25 ml flask was added 4 (225 mg, 1 mmol) and 10 ml CH₃CN. HClgas was passed through the solution for 3 h, before the solvent wasevaporated under reduced pressure. The residue was dissolved in 10 mLdistilled water and treated with ammonia water solution to generate awhite precipitate. The precipitate was collected by filtration to afford143 mg (62%) of 5 as a white solid: ¹H NMR (DMSO-d₆) δ 1.75 (m, 4 H),2.48 (s, 3 H), 2.69 (m, 2 H), 2.83 (m, 2 H), 12.16 (s, 1 H). Anal.(C₁₁H₁₂N₂OS) m/z (ESI) 220.067811 [M]⁺.

4-Chloro-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine (6).To a 50 mL flask was added 5 (220 mg, 1 mmol) and 5 mL POCl₃. Theresulting mixture was refluxed for 2 h, and the solvent was removedunder reduced pressure to afford a dark residue. To this was added 30 mLof CHCl₃ and 300 mg of silica gel. The solvent was evaporated to afforda plug. Column chromatography of the plug with hexane: acetylacetate=20:1 as eluent afford 192 g (81%) of 6 as a yellow solid: ¹H NMR(DMSO-d₆) δ 1.82 (m, 4 H), 2.63 (s, 3 H), 2.84 (m, 2 H), 2.96 (m, 2 H).

N-(4-methoxyphenyl)-N,2-dimethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-amine(AAG101). To a 50 mL flask was added 6 (119 mg, 0.5 mmol), 7 (77 mg,0.55 mmol) and 5 mL BuOH. To this solution was added 2 drops ofconcentrate HCl solution and the mixture was refluxed. TLC indicated thedisappearance of starting material 6, the solvent was removed underreduced pressure. To the residue obtained was added silica gel and MeOHand the solvent removed to make a plug. This plug was separated bycolumn chromatography to give 110 g (65%) of 8 as a white powder: mp108-109° C.; R_(f) 0.36 (Hexane/EtOAC 3:1); ¹H NMR (DMSO-d₆) δ 2.14 (s,3 H), 2.45 (s, 3 H), 3.43 (s, 3 H), 3.81 (s, 3 H), 4.55 (s, 1 H), 7.04(d, 2 H, J=2.8 Hz), 7.25 (d, 2 H, J=2.8 Hz). Anal. (C₁₉H₂₁N₃OS) m/z(ESI) 340.1484 [M]⁺.

Synthesis of AAG102

Chemistry:

The Gewald reaction of 1, 2 and 3 afforded bicyclic 4 (Scheme 3).Condensation of 4 and 9 afforded 10, which was converted to the 4-chloroanalogue 11 by treating with POCl₃. The 4-chloro compound 11 reactedwith N-methyl aniline 7 and a trace amount of HCl in BuOH to givecompound AAG102.

Experimental Section for Scheme 4

2-amino-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (10).To a 50 mL flask was added 4 (225 mg, 1 mmol), 9 (452 mg, 4 mmol) inDMSO₂ (500 mg) was heated at 150° C. for 2 h. The mixture was cooled toroom temperature. 15 mL water was added and ammonium hydroxide was usedto neutralize the suspension. The brown solid was obtained byfiltration. Washed with water and dried. The solid was dissolved inmethanol and silica gel was added. A dry silica gel plug obtained afterevaporation. The plug was then loaded on the column and washed with 5%methanol in chloroform. The fractions containing the desired product(TLC) were pooled and evaporated to afford 132 mg (60%) of 10 as a whitesolid: ¹H NMR (DMSO-d₆) δ 1.71 (m, 4 H), 2.60 (m, 2 H), 2.72 (m, 2 H),6.38 (s, 2 H), 10.72 (s, 1 H). Anal. calcd for C₁₀H₁₁N₃OS: C, 54.28; H,5.01; N, 18.99; 0, 7.23; S, 14.49. Found: C, 53.45; H, 5.06; N, 18.26;S, 14.02.

4-Chloro-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-2-amine (11).To a 50 mL flask was added 10 (221 mg, 1 mmol) and 10 mL POCl₃. Theresulting mixture was refluxed for 2 h, and the solvent was removedunder reduced pressure to afford a dark residue. To this was added 30 mLof CHCl₃ and 300 mg of silica gel. The solvent was evaporated to afforda plug. Column chromatography of the plug with hexane: acetylacetate=20:1 as eluent afford 105 mg (44%) of 11 as a yellow solid:

N⁴-(4-methoxyphenyl)-N⁴-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-2,4-diamine(AAG102). To a 50 mL flask was added 11 (119 mg, 0.5 mmol), 7 (77 mg,0.55 mmol) and 5 mL BuOH. To this solution was added 2 drops ofconcentrate HCl solution and the mixture was refluxed. TLC indicated thedisappearance of starting material 11, the solvent was removed underreduced pressure. To the residue obtained was added silica gel and MeOHand the solvent removed to make a plug. This plug was separated bycolumn chromatography to give 105 mg (62%) of 12 as a white powder: ¹HNMR (DMSO-d₆) δ 1.38 (m, 2 H), 1.52 (m, 2 H), 1.68 (m, 2 H), 2.07 (m, 2H), 3.69 (s, 3 H), 6.37 (s, 2 H), 6.84 (m, 4 H). Anal. (C₁₈H₂₀N₄OS) m/z(ESI) 341.1436 [M+1]⁺

Chemical Discussion:

The synthesis of target compounds commenced from commercially available2-amino-6-chloropyrimidin-4(3H)-one (1) using a method reported byTaylor et al.¹ to obtain 2 in 46% yield (Scheme 5). A thermal FisherIndole-cyclization of 2 and cyclohexanone in diphenyl ether furnishedthe tricyclic scaffold 3. The partially saturated ring in 3 was oxidizedusing 10% Pd/C to provide 4 in 57% yield. Pivaloyl protection of 4 gave5, and subsequent chlorination afforded the synthetic intermediate 6.Compound 6 was treated with various substituted anilines in isopropanaland three drops of concentrated HCl at reflux to provide the penultimatecompounds 7-11 in yields of 37 to 80%. The pivaloyl group in 7-11 wasremoved by a basic hydrolysis of the 2-amide linkage in these compoundsusing 1N NaOH to give the desired target compounds-AAG103-AAG107 inyields ranging from 64-93%. AAG107 was also synthesized by treating 6with 3-methoxy-N-methyl aniline, in n-butanol and three drops of conc.HCl at reflux, to afford 16 in a yield of 53%

Experimental Section for Scheme 5:

2-Amino-6-hydrazinopyrimidin-4(3H)-one (2). A stirred suspension of 15.0g (103 mmol) of 1 in 250 mL water was added 12 g (375 mmol) of anhydroushydrazine, and the mixture was heated at reflux for 3 h. The resultingclear solution was cooled, and the precipitate which separated wascollected by filtration, washed with water followed by ethanol and driedto give 6.6 g (46%) of white solid. mp decomposes at 313° C. (Literaturemp 314-315° C., see Taylor, E. C.; Cocuzza, J. A. Synthesis andProperties of 7-Azaxanthopterin. J. Org. Chem. 1979, 44, 1125-1128).

2-Amino-3,5,6,7,8,9-hexahydro-4H-pyrimido[4,5-b]indol-4-one (3). Amixture of 2 (350 mg, 2.5 mmol) and cyclohexanone (245 mg, 2.5 mmol) wasstirred and heated at 120-130° C. in an oil bath overnight and thenunder reflux at 250° C. for 3 hours. After cooling to room temperature,hexane (50 mL) was added and the precipitated solid was collected byfiltration. The solid was dried over P₂O₅, dissolved in methanol andadded silica (three times the weight of solid), following which thesolvent was removed under reduced pressure to obtain a dry plug. Theplug was loaded on top of a column packed with chloroform. The weight ofsilica in the column was thirty times the weight of the plug. Flashchromatography using 20% methanol in chloroform afforded 82% of yellowsolid. TLC R_(f) 0.35 (chloroform-methanol 5:1); mp decomposes at 338°C.; ¹H NMR (DMSO-d⁶) δ 1.64-1.69 (m, 4 H, 6-CH₂, 7—CH₂), 2.42 (m, 2 H,5-CH₂), 2.53 (m, 2 H, 8-CH₂), 5.88 (s, 2 H, 2-NH₂, exch), 10.02 (s, 1 H,3-NH, exch), 10.50 (s, 1 H, 9-NH, exch). Anal. Calculated (C₁₀H₁₂N₄O.0.4 H₂O): C, 56.80; H, 6.10; N, 26.49. Found: C, 56.90; H, 6.06; N,26.49.

2-Amino-3,9-dihydro-4H-pyrimido[4,5-b]indol-4-one (4). A mixture of 3(50 mg, 0.24) and 10% Pd/C (24 mg) in Ph₂O (5 mL) was heated to 250° C.for 3 hours. The reaction mixture was cooled to room temperature and DMF(20 mL) was added to dissolve the product. The catalyst was filtered offthrough celite and washed with DMF to give a solution which wasevaporated to give solid residue. Hexane (25 mL) was added to theresidue and the solid was filtered. The solid was dissolved in DMF (5mL) and silica gel (1 g) was added. Solvent was removed under reducedpressure to afford plug, which was loaded on top of a silica gel columnpacked with chloroform, having silica, thirty times by weight of theplug, and eluted with 20% methanol in chloroform to afford 28 mg of 4 asa pale white solid in 57% yield. TLC R_(f) 0.32 (chloroform-methanol5:1); mp>340° C.; ¹H NMR (DMSO-d⁶) δ 6.51 (s, 2 H, NH₂), 7.04 (m, 2 H,Ar—H), 7.24 (m, 1 H, Ar—H), 7.7 (m, 1 H, Ar—H), 10.47 (s, 1 H, 3-NH,exch), 11.35 (s, 1 H, 9-NH, exch).

2,2-Dimethyl-N-(4-oxo-4,9-dihydro-3H-pyrimido[4,5-b]indol-2-yl)propanamide(5). Compound 4 (50 mg, 0.25 mmol), trimethyl acetic anhydride (6 g, 32mmol), DMAP (16 mg, 0.13 mmol), and triethylamine (101 mg, 1 mmol) weredissolved in 8 mL of DMF. The mixture was heated at 60° C. for 40 hours.The DMF and trimethyl acetic anhydride were removed under reducedpressure using oil pump. The residue thus obtained was dissolved inmethanol, and was added 3 g of silica gel. The solvent was removed underreduced pressure to afford a dry plug. The plug was loaded on top of asilica gel column packed with chloroform, having silica, twenty times byweight of the plug, and eluted with 1% methanol in chloroform to obtain52 mg of 5 as a yellow solid in 73% yield. TLC R_(f) 0.67(chloroform-methanol 5:1); mp decomposes at 322° C.; ¹H NMR (DMSO-d⁶) δ1.30 (s, 9 H, C(CH₃)₃), 7.17-7.94 (m, 4 H, Ar—H), 11.12 (s, 1 H, NH,exch), 11.85 (s, 1 H, NH, exch), 12.01 (s, 1 H, NH, exch). Anal.Calculated (C₁₅H₁₆N₄O₂. 0.3 H₂O): C, 62.18; H, 5.77; N, 19.33. Found: C,62.24; H, 5.83; N, 18.98.

N-(4-chloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide (6).In a 50 mL R.B.F. was placed 5 (56 mg, 0.17 mmol) and phosphoryltrichloride (15 mL). The mixture was stirred and heated at reflux for 4hours. The phosphoryl trichloride was evaporated under reduced pressureusing a vacuum aspirator. The resulting residue was cooled in an ice andwater mixture, and neutralized with ammonium hydroxide solution to yielda precipitate that was filtered and dried over P₂O₅. The filterate wasextracted with chloroform and dried over sodium sulfate. The dryprecipitate and filterate were combined and evaporated to provide 35 mgof 6 as a brown solid in 59% yield. Compound 6 was used without furtherpurification for the subsequent step. TLC R_(f) 0.65(chloroform-methanol 10:1); mp 234° C.; ¹H NMR (DMSO-d⁶) δ 1.24 (s, 9 H,C(CH₃)₃), 7.36-8.17 (m, 4 H, Ar—H), 10.25 (s, 1 H, 2-NH, exch), 12.55(s, 1 H, 9-NH, exch). Anal. Calculated (C₁₅H₁₅ClN₄O. 0.15 H₂O): C,58.98; H, 5.04; N, 18.34; Cl, 11.60. Found: C, 59.00; H, 5.09; N, 17.97;Cl, 11.83.

General procedure for the synthesis of N-{4-[substituted phenylamino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamides 7-11.Compound 6 (1 equivalent) was dissolved in isopropanol, and to thissolution was added a substituted aniline (7.5 equivalents). Three dropsof conc. HCl were added, and the resulting mixture was stirred andheated at 110° C. from 1 to 5 days, depending upon the aniline used. Forwork up of this reaction, the solvent was removed under reducedpressure, the mixture was basified with ammonia in methanol, dissolvedin methanol, added silica, and the solvent was evaporated to give a dryplug. The plug was loaded on top of a silica gel column packed withchloroform and having silica, fifty times by weight of the plug, andeluted with 0.2% methanol in chloroform to obtain compounds 7-11 inyields of 37-80%.

N-{4-[(3-bromophenyl)amino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide(7). Using the general procedure described above, the reaction of 6 (181mg, 0.59 mmol) and 3-bromoaniline (771 mg, 4.48 mmol), was run for 26hours, to provide 209 mg of 7 as a white solid in 80% yield. TLC R_(f)0.28 (chloroform-methanol 20:1); mp 286.7-287.5° C.; ¹H NMR (DMSO-d⁶) δ1.27 (s, 9 H, C(CH₃)₃), 7.19-7.49 (m, 4 H, Ar—H), 8.18-8.46 (m, 4 H,Ar—H), 8.79 (s, 1 H, 4-NH, exch), 9.64 (s, 1 H, 2-NH, exch), 11.89 (s, 1H, 9-NH, exch). Anal. Calculated (C₂₁H₂₀BrN₅O): C, 57.54; H, 4.59; N,15.97; Br, 18.22. Found: C, 57.34; H, 4.65; N, 15.80; Br, 17.96.

N-{4-[(3-methoxyphenyl)amino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide(8). Using the general procedure described above, the reaction of 6 (80mg, 0.26 mmol) and 4-methoxyaniline (244 mg, 1.98 mmol) was run for 18hours, to provide 52 mg of 8 as a white solid in 51% yield. TLC R_(f)0.55 (chloroform-methanol 15:1); mp 266.8-267° C.; ¹H NMR (DMSO-d⁶) δ1.23 (s, 9 H, C(CH₃)₃), 3.75 (s, 3 H, OCH₃), 6.88-8.26 (m, 8 H, Ar—H),8.55 (s, 1 H, 4-NH, exch), 9.41 (s, 1 H, 2-NH, exch), 11.79 (s, 1 H,9-NH, exch). Anal. Calculated (C₂₂H₂₃N₅O₂. 0.4 H₂O): C, 66.61; H, 6.04;N, 17.65. Found: C, 66.61; H, 6.00; N, 17.37.

N-{4-[(3-methoxyphenyl)(methyl)amino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide(9). Using the general procedure described above, the reaction of 6 (180mg, 0.59 mmol) and 4-methoxy-N-methylaniline (612 mg, 4.46 mmol) was runfor 72 hours, to provide 109 mg of 9 as brown crystals in 45% yield. TLCR_(f) 0.62 (chloroform-methanol 15:1); mp 248.2-249° C.; ¹H NMR(DMSO-d⁶) δ 1.26 (s, 9 H, C(CH₃)₃), 3.61 (s, 3 H, OCH₃), 3.74 (s, 3 H,NCH₃), 5.76-7.29 (m, 8H, Ar—H), 9.49 (s, 1 H, 2-NH, exch), 11.81 (s, 1H, 9-NH, exch). Anal. Calculated (C₂₃H₂₅N₅O₂. 0.55 CH₃OH): C, 67.16; H,6.51; N, 16.63. Found: C, 67.33; H, 6.49; N, 16.28.

N-{4-[(4-methoxyphenyl)amino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide(10). Using the general procedure described above, the reaction of 6(160 mg, 0.52 mmol) and 3-methoxyaniline (488 mg, 3.96 mmol) was run for36 hours, to provide 107 mg of 10 as an off white solid in 52% yield.TLC R_(f) 0.64 (chloroform-methanol 15:1); mp 219.4-220.2° C.; ¹H NMR(DMSO-d⁶) δ 1.23 (s, 9 H, C(CH₃)₃), 3.8 (s, 3 H, OCH₃), 6.57-8.31 (m, 8H, Ar—H), 8.62 (s, 1 H, 4-NH, exch), 9.59 (s, 1 H, 2-NH, exch), 11.85(s, 1 H, 9-NH, exch). Anal. Calculated (C₂₃H₂₅N₅O₂. 0.55 CH₃OH): C,67.16; H, 6.51; N, 16.63. Found: C, 67.33; H, 6.49; N, 16.28.

N-{4-[(4-methoxyphenyl)(methyl)amino]-9H-pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide(11). Using the general procedure described above, the reaction of 6(180 mg, 0.59 mmol) and 4-methoxy-N-methylaniline (612 mg, 4.46 mmol)was run for 120 hours, to provide 88 mg of 11 as a brown solid in 37%yield. TLC R_(f) 0.57 (chloroform-methanol 15:1); mp 288.5-289.4° C.;^(i)H NMR (DMSO-d⁶) δ 1.26 (s, 9 H, C(CH₃)₃,) 3.60 (s, 3 H, OCH₃), 3.69(s, 3 H, NCH₃), 5.87-7.33 (m, 8 H, Ar—H), 9.58 (s, 1 H, 2-NH, exch),11.88 (s, 1 H, 9-NH, exch).

General procedure for the synthesis ofN⁴-substituted-9H-pyrimido[4,5-b]indole-2,4-diamine AAG103-AAG107.Compounds 7-11 were dissolved individually in isopropanol. About 4 mL of1 N NaOH was added to this solution and the resulting mixture wasstirred and heated at 110° C. from 14 hours. For work up of thisreaction, the solvent was removed under reduced pressure, and theresidue was dried over P₂O₅. The dry residue was dissolved in methanol,added silica, and the solvent was evaporated to give a dry plug. Theplug was loaded on top of a silica gel column (having silica, fifteentimes by weight of the plug and packed with chloroform), and eluted with1% methanol in chloroform to obtain compounds AAG103-AAG107respectively. These compounds were washed with nonpolar solvents(hexane, diethyl ether) before being sent for elemental analysis. Theyields ranged from 64-93%.

N⁴-(3-bromophenyl)-9H-pyrimido[4,5-b]indole-2,4-diamine AAG103. Usingthe general procedure described above, the reaction of 7 (209 mg, 0.47mmol) and 1 N NaOH provided 128 mg of AAG103 as a white solid in 76%yield. TLC R_(f) 0.36 (chloroform-methanol 15:1); mp 233.6° C.; ¹H NMR(DMSO-d⁶) δ 6.26 (s, 2 H, NH₂, exch), 7.09-8.09 (m, 8 H, Ar—H), 8.44 (s,1 H, 4-NH, exch), 11.32 (s, 1 H, 9-NH, exch). Anal. Calculated(C₁₆H₁₂BrN₅): C, 54.25; H, 3.41; N, 19.77; Br, 22.55. Found: C, 54.38;H, 3.48; N, 19.56; Br, 22.29.

N⁴-(3-methoxyphenyl)-9H-pyrimido[4,5-b]indole-2,4-diamine AAG104. Usingthe general procedure described above, the reaction of 8 (90 mg, 0.23mmol) and 1 N NaOH provided 65 mg of AAG104 as a transparent solid in92% yield. TLC R_(f) 0.34 (chloroform-methanol 15:1); mp 225.2-225.6°C.; ¹H NMR (DMSO-d⁶) δ 3.74 (s, 3 H, OCH₃), 6.05 (s, 2 H, NH₂, exch),6.88-8.07 (m, 8 H, Ar—H), 8.16 (s, 1 H, 4-NH, exch), 11.20 (s, 1 H,9-NH, exch). Anal. Calculated (C₁₇H₁₅N₅O. 0.15 (C₂H₅)₂O): C, 66.79; H,5.25; N, 22.13. Found: C, 66.77; H, 5.08; N, 22.13.

N⁴-(3-methoxyphenyl)-N⁴-methyl-9H-pyrimido[4,5-b]indole-2,4-diamineAAG105. Using the general procedure described above, the reaction of 9(90 mg, 0.22 mmol) and 1 N NaOH provided 66 mg of AAG105as a white solidin 93% yield. TLC R_(f) 0.27 (chloroform-methanol 15:1); mp 245.3-245.7°C.; ¹H NMR (DMSO-d⁶) δ 3.48 (s, 3 H, OCH₃), 3.71 (s, 3 H, NCH₃), 6.20(s, 2 H, NH₂, exch), 5.75-7.15 (m, 8 H, Ar—H), 11.22 (s, 1 H, 9-NH,exch). Anal. Calculated (C₁₈H₁₇N₅O. 0.17 H₂O): C, 67.05; H, 5.42; N,21.72. Found: C, 67.06; H, 5.37; N, 21.56.

N⁴-(4-methoxyphenyl)-9H-pyrimido[4,5-b]indole-2,4-diamine AAG106. Usingthe general procedure described above, the reaction of 10 (80 mg, 0.20mmol) and 1 N NaOH provided 40 mg of AAG106 as an off-white solid in 64%yield. TLC R_(f) 0.30 (chloroform-methanol 15:1); mp 208.9-209.1° C.; ¹HNMR (DMSO-d6) δ 3.76 (s, 3 H, OCH₃), 6.19 (s, 2 H, NH₂, exch), 7.09-8.05(m, 8 H, Ar—H), 8.26 (s, 1 H, 4-NH, exch), 11.27 (s, 1 H, 9-NH, exch).Anal. Calculated (C₁₇H₁₅N₅O. 0.35 CH₃OH): C, 65.83; H, 5.22; N, 22.12.Found: C, 66.08; H, 5.22; N, 21.81.

N⁴-(4-methoxyphenyl)-N⁴-methyl-9H-pyrimido[4,5-b]indole-2,4-diamineAAG107. Using the general procedure described above, the reaction of 11(100 mg, 0.24 mmol) and 1 N NaOH provided 58 mg of AAG107 as a whitesolid in 73% yield. Alternatively, AAG107 can also be synthesized bytreating 6 (90 mg, 0.3 mmol) with 3-methoxy-N-methylaniline (306 mg,2.23 mmol) in presence of 3 drops HCl and solvent n-butanol under refluxconditions for 14 hours. The yield from 6 to AAG107 using this method is53%. TLC R_(f) 0.34 (chloroform-methanol 15:1); mp 233.6° C.; ¹H NMR(DMSO-d⁶) δ 3.55 (s, 3 H, OCH₃), 3.58 (s, 3 H, NCH₃), 6.19 (s, 2 H, NH₂,exch), 5.87-7.18 (m, 8 H, Ar—H), 11.27 (s, 1 H, 9-NH, exch).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A method of treating a patient diagnosed withcancer comprising administering to the patient a therapeuticallyeffective amount of a compound of Formula I, or a pharmaceuticallyacceptable salt of the compound of Formula I:

wherein both B and C rings may be completely or partially saturated withrespect to bonds 4a-8a, 5-6 and 7-8; the C ring may have an R₄ attachedto the C ring at positions 5, 6, 7 or 8, or a combination of one or moreof these positions depending on the saturation level of the C ring andwherein R₄ may be the same or different when attached to a plurality ofthe 5, 6, 7, or 8 positions of the C ring; R₁and R₂ each independentlyis one of (a) a hydrogen (H), (b) an alkyl having from one to ten carbonatoms and having a straight or branched configuration, and wherein thealkyl is partially or completely saturated, or a substituted alkylhaving from one to ten carbon atoms, (c) a cycloalkyl having from threeto ten carbon atoms, or a substituted cycloalkyl having from three toten carbon atoms, (d) an alkylcycloalkyl, or a substitutedalkylcycloalkyl, (e) an aryl, or a substituted aryl, (f) an alkylaryl,or a substituted alkylaryl (g) a heteroaryl, or a substitutedheteroaryl, (h) an alkylheteroaryl, or a substituted alkylheteroaryl,(i) an aromatic, or a substituted aromatic, and (j) a heteroaromatic, ora substituted heteroaromatic, and wherein each substituent of any saidsubstituted group is the same or different and is selected from thegroup consisting of a straight or branched alkyl, alkenyl, or alkynyl, acyclic or alicyclic group having from one to six carbon atoms, aheterocyclic group having from one to six carbon atoms, an alkoxy group,an aryloxy group, an alkyloxyaryloxy group, an aryl group, an amine, ahalogen, a phenol, a naphthalene, a piperidine, a pyrrole, a ketone, amethylalkyl ketone, and a trifluoromethyl ketone, and wherein each ofsaid substituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; R₁ and R₂ arethe same or different with the exception that R₁ and R₂ may not each behydrogen at the same time; R₃ is one of (a) a hydrogen (H), (b) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (c) an NH₂, (d) an NHR₇, (e) an NR₇R₈, (f) an OH, (g) an OR,(h) an SH, and (i) an SR, and wherein R is one of R₁, and wherein R₇ andR₈ may be the same or different and is one of R₁; R₄ is one of (a) R₁,(b) a halogen, (c) a mono-, di-, tri- or tetra-substituted alkyl, and(d) an alkyloxy, and wherein R₁ is H or a lower alkyl and R₂ is H or alower alkyl then R₄ is one of (a) a NR₆R₇, (b) a SR₆, (c) a OR₆, and (d)a CHR₆R₇, wherein R₆ and R₇ are one of R₁ and R₂, and R₆ and R₇ are thesame when said C ring is completely or partially saturated; X is asulfur (S); Y is one of (a) a nitrogen (N), (b) an oxygen (O), (c) asulfur (S), and (d) a CR₆, wherein R₆ is one of R₁ and R₃, and whereinwhen Y is ₀ or S then either R₁ or R₂ is absent; wherein when said Cring is partially saturated and X is a sulfur (S) and Y is a sulfur (S)and R₃ is a hydrogen (H) or a methyl, then (a) R₁ is not a methyl whenR₂ is absent; and (b) R₂ is not a methyl when R₁ is absent; wherein whensaid C ring is partially saturated and X is a sulfur (S) and Y is anoxygen (O) and R₃ is a methyl, then (a) R₁ is not an ethyl or a propylwhen R₂ is absent; and (b) R₂ is not an ethyl or a propyl when R₁ isabsent; wherein when said C ring is partially saturated and X is asulfur (S) and Y is a nitrogen (N) and R₃ is a methyl, then (a) R₁ isnot a hydrogen (H) when R₂ is a butyl; and (b) R₂ is not a hydrogen (H)when R₁ is a butyl; and (c) R₁ and R₂ are not both an ethyl; whereinwhen X is a sulfur (S) and Y is a nitrogen (N) and R₃ is a hydrogen (H),then neither R₁ nor R₂ is a substituted phenyl group; for achieving atherapeutic response.